So the aim of the method in our manuscript is to provide comparative analysis of signaling pathways in myeloid cells using human and mouse systems. It helps define which signaling pathway is involved downstream of selected pattern recognition receptors. So this method is simple to implement.
It relies on primary cell preparations and validated reference compounds, which is essential for the elucidation of signaling mechanisms. Demonstrating the procedure will be Ratiba Touil and Adeline Unterreiner, respectively scientist and research associate in our group. In a laminar flow hood set out a sterile pair of scissors, a one liter beaker, and a plastic bag.
Place the previously obtained buffy coat into the beaker and use the scissors to carefully open it. Using a 25 mm pipette add 100 ml of PBS supplemented with 2 millimolar of EDTA. Mix by slowly pipetting up and down.
And then transfer 25 ml of the diluted buffy coat into six 50 ml conical centrifuge tubes that have each been prefilled with 15 ml of polysaccharide based density gradient. Centrifuge the tubes for 20 minutes at 800 g with moderate acceleration without the break to allow for separation of cells based on their densities. After centrifugation three layers will be visible, a pellet containing red blood cells and granulocytes, an upper layer made of plasma, and a white ring containing peripheral blood mononuclear cells.
Use a 10 ml pipette to harvest the PBMC rings and transfer them into a new 50 ml tube. Top up with PBS and EDTA. Perform three successive washes with decreasing centrifugation time and speed as outlined in the text protocol.
After the final wash, resuspend the pellet in 25 ml of ice cold lysis buffer to lyse the red blood cells by osmotic pressure. Incubate the solution at room temperature until it becomes clear. Then add 25 ml of separation buffer to stop the reaction.
Centrifuge at 150 g for eight minutes to wash the solution. Pour off the supernatant and resuspend the pellet with separation buffer. After counting the cells, dilute them in culture medium down to 12, 500 cells per well.
Distribute 30 ml of this cell suspension into each well of a 384 well plate. Next add 15 microliters of four times concentrated compound solutions to each well and preincubate the plate at 37 degrees Celsius with 5%carbon dioxide for one hour. If desired, add either LPS to final concentration of 1 ng per ml or depleted Zymosan to a final concentration of 100 mcg per ml.
Then incubate the plate overnight at 37 degrees Celsius with 5%carbon dioxide. The next day take 10 microliters of the supernatant to measure the secreted TNF alpha levels. To begin the serial dilution, dilute the MLT-827 stock solution with medium to reach a concentration of 8 micromolar in one go.
Perform a six-step 1:5 serial dilution using medium with 0.08%DMSO. To prepare for single dose testings, dilute the MLT-827, AFN700, and compound 11 stock solutions with medium to reach a concentration of 4 micromolar in one go. First, add 2 ml of sterile endotoxin-free water to 10 mg of depleted Zymosan.
Vortex this stock solution to homogenize it. Aliquot the solution and then store the aliquots at 20 degrees Celsius. In this study human PBMCs and mouse spleen cells are stimulated with either depleted Zymosan, a Dectin-1 agonist, or lipopolysaccharide, a TL4 agonist.
NTNF alpha release in the supernatant is measured after 20 hours. In both the human and mouse assays the MLT-827 compound selectively blocks TNF alpha production driven by the Dectin-1 pathway but not by the TLR4 pathway. In monocytes stimulated with LPS, production of TNF alpha is almost completely abrogated by AFN700 but it's not sensitive to Cpd11, which is consistent with the TLR4 pathway's dependency on NF-kappa B activity and its independency on SIC activity.
In contrast TNF alpha production driven by Dectin-1 and MODCs shows sensitivity to MLT-827, AFN700, and Cpd11. This provides further evidence for the involvement of a SIC CBM signaling cascade in the Dectin-1 pathway. Production of IL-1 beta, IL-6, and IL-23 is also sensitive to the three inhibitors, indicating regulatory mechanisms similar to TNF alpha.
However, the limited effect seen on the production of IL-8 suggests that this cytokine has a distinct regulatory mechanism. Raising Trehalose-dibehenate concentrations above 50 mcg per ml leads to production of TNF alpha, IL-6, and IL-1 beta, which relied on MALT1 paracaspase activity according to the blocking effect of MLT-827. Similar results were seen when challenging the MODCs with increasing concentrations of depleted Zymosan to stimulate Dectin-1.
This method made it possible to show that the paracaspase MALT1 selectively regulates C-type lectin-like receptor signaling. It is essential to use well-characterized reference compounds. Pay attention to the procedure for the dilution of stock solutions.
This is critical for compound with limited solubility.
